Several murine studies have suggested that the strength of the initial T cell response to a new, infecting virus is influenced by a preexisting T cell repertoire, induced by previous infections with other viruses. Each subsequent exposure to a new virus is thought to alter the memory T cell repertoire and to selectively expand crossreactive memory T cells (Immunity 1999; 11: 733-42). To examine this hypothesis in humans, we searched for T cells with crossreactivity against hepatitis C virus and other viruses. Indeed, after three rounds of in vitro stimulation with peptide in the presence of various cytokines, we were able to expand CTL specific for an HCV epitope not only from the blood of HCV infected patients, but also from the blood of 9/15 (60%) HCV negative blood donors. A databank search identified a highly homologous peptide from the influenza A virus that bound to the HLA-A2 molecule with comparable, although low affinity. Direct ex vivo analysis by IFNg-Elispot demonstrated that 4 of the 9 HCV negative blood donors with, but none without specific CTL responses against the HCV epitope, recognized the influenza peptide. Recognition of either peptide could be competed by cold target cells loaded with the other peptide and HCV specific T cells did also recognize target cells that were infected with influenza A virus and processed the influenza peptide endogenously. To demonstrate in vivo that influenza A virus infection can induce HCV specific CTL, we infected HLA-A2 transgenic mice with influenza A virus and showed that both effector and memory T cells, induced by the influenza A virus, recognized the HCV peptide. These results demonstrate that host responses to an infectious agent are influenced by cross-reactive memory cells induced by past exposure to heterologous viruses, a finding, that may be of clinical relevance for vaccine development.In a second study, we induced HCV specific T cell responses with an oral immunization strategy in mice. We employed live attenuated Salmonella typhimurium transformed with a plasmid carrying the HCV NS3 coding region to deliver the cDNA directly to large numbers of professional antigen presenting cells in the gut associated lymphoid tissue. Induction of HCV-specific CD8+ T cells was demonstrated by IFN-g ELISPOT and CTL assay. To investigate the in vivo function of HCV-specific CTL, we challenged mice with wildtype or HCV NS3 expressing vaccinia virus. While vaccinia titers after infection with wildtype VV were not different, the titers after challenge with HCV NS3-VV were significantly lower in mice immunized with salmonella-NS3 than in mice immunized with salmonella without HCV antigen. This in vivo protection lasted for at least 10 months after immunization and thus, oral immunization with live, attenuated Salmonella typhimurium as a carrier for HCV-cDNA is a potent strategy to induce long-lasting HCV-specific CTL responses.